P-091: EFFECTS OF CARICA PAPAYA LEAF EXTRACTS IN TRANSCRIPTIONAL REGULATION OF FETAL HEMOGLOBIN
Authors: MENDES M., CANTEIRO B., DELGADINHO M., OLIVEIRA K., GINETE C., GOMES M., RIBEIRO E., BRITO M., GOMES A.
Journal: HemaSphere (2022)

Purpose: Sickle cell disease (SCD) is one of the most common human genetic disorders, which is caused by a single point mutation (Glu6Val) on the HBB gene. Currently, one of the treatments for this global health problem involves induction of fetal hemoglobin (HbF). There are some drugs on the market that pharmacologically induce HbF, namely Hydroxyurea (HU), however, their safety concerns and the expensive cost in low- and middle-income countries limit their use. In this context, it is essential to study novel fetal hemoglobin-inducing compounds that have fewer adverse effects and are widely available, such as natural compounds. Therefore, the main aim of this work was to evaluate the effects of Carica Papaya methanolic leaf extracts (CPMLE) in HbF reactivation. Materials and methods: In order to achieve these goals, gene expression studies on globin and HbF regulators/silencers genes were performed using three biological replicates in K562 cells. The cells were exposed for 24 hours to three concentrations of CPMLE (0,5; 50 and 100 μg/ml), and to 25 μg/ml of HU, which was used as a positive control. Exposed cells and controls were harvested, and parameters such as cell proliferation and cell viability were microscopically evaluated. Variation in gene expression after CPMLE exposition was quantified from total RNA by quantitative Real Time PCR. The studied genes were α, β and γ-globin genes, as well as the HbF regulators genes MYB, KLF1, BCL11A and BGLT3, and GAPDH was used as a reference gene. Results: The proliferation rates were calculated as the ratio between the value at 24h and the initial number of cells (1X105 cells/well). The results for the CPMLE concentrations of 0,5; 50 and 100 μg/ml were of 2,12; 2,48 and 2,15, respectively, while for control (cells non-treated) the value was 2,35. The percentages obtained for the viability, as assessed by trypan blue staining, were of 90,02% in control cells, 94,33%; 89,22% and 84,42% for the CPMLE concentrations of 0,5; 50 and 100 μg/ml, respectively. Altogether, these results indicate that some concentrations of CPMLE affect the proliferation and viability of K562 cells, although no cytotoxic effects were observed. Transcriptional analysis demonstrated that all CPMLE concentrations repress BCL11A expression and increase expression of HBA, HBB, MYB and KLF1, relative to control cells, which is in line with the HU results observed for each gene. Additionally, an overexpression of γ-globin and BGLT3 genes was observed upon incubation with 0,5 μg/ml of CPMLE. Thus, the results observed for the BGLT3 gene where distinct between CPMLE and HU. For the other CPMLE concentrations, there was a reduction in the expression of the same genes. Conclusion: Overall, this preliminary study suggests that CPMLE can modulate the expression of HbF and regulator genes, thus potentially constituting an effective approach for treatment of SCD. Acknowledgments This project was supported by Instituto Politécnico de Lisboa under the grant IDI&CA-IPL/2021/EpiCa/ESTeSL and partially supported by FCT/MCTES (UIDB/05608/2020 and UIDP/05608/2020). The authors are grateful to Fernando Nunes for kindly providing the Carica Papaya leaves. The authors do not declare any conflict of interest